Novel Cytokine Containing Compositions

ABSTRACT

The invention is directed to novel cellular factor-containing solution compositions (referred to herein as “CFS” compositions), including novel sustained-release cellular factor-containing solution compositions (referred to herein as “SR-CFS” compositions), methods of making such novel compositions and uses thereof.

FIELD OF THE INVENTION

The field of the invention is directed to novel cellularfactor-containing solution compositions (referred to herein as “CFS”compositions), including novel sustained-release cellularfactor-containing solution compositions (referred to herein as “SR-CFS”compositions), methods of making such novel compositions and usesthereof.

BACKGROUND OF THE INVENTION

Many individual cytokines and growth factors have been evaluated fortheir therapeutic utility in the treatment of many varied diseases,disorders and injuries. Unfortunately, the results have been onlypartially encouraging. For example, PDGF-BB has proven to be useful inthe treatment of diabetic foot ulcers; GM-CSF is marketed in Europe forboth venous ulcers and diabetic foot ulcers; and HGH (human growthhormone) is marketed in the US for pediatric burns. Failures includeBDNF, CNTF and IGF-1 which have all been evaluated in clinical trialsdesigned to test their efficacy in treating ALS, each with disappointingresults; TGFβ2 was unsuccessful in a phase 2 study for venous ulcers;and IGF-1 and PDGF combination therapy was unsuccessful in diabetic footulcers.

While is not clear why so many of these individual cytokines and growthfactors have failed in the clinic, one theory is that the proteins werebeing administered in doses that were not physiologic, i.e. very highdoses compared to the physiologic levels normally found in vivo. Also,because of the complex interaction between cytokines and growth factorsin a given physiological niche, the application of just one factor,especially one at abnormally high levels, cannot recreate thephysiological niche and may, in fact, grossly disturb its delicatebalance.

Compounding their limited success in the clinic, cytokines and growthfactors and other protein-based therapeutics are typically moredifficult to administer to patients than other pharmaceuticals. Becausethe efficacy of a protein is related to its shape, protein-basedtherapeutics cannot be subjected to conditions that could cause theunfolding, or denaturing, of the protein or proteins contained therein.Consequently, special care is necessary in the preparation, storage, andadministration of protein-based therapeutics.

In addition to avoiding any denaturation of the protein, it is oftendesirable to be able to control the amount of the protein administeredto a patient over time. This helps to avoid protein concentrationswithin the patient that are undesirably high or low or that fluctuatetoo much from a desired level, and instead helps maintain a steady levelof the therapeutic in the patient. To address this, sustained-releaseformulations for many therapeutics, including protein-basedtherapeutics, have been or are currently in development.Sustained-release protein-based therapeutics can be administered by avariety of methods, including but not limited to oral delivery oftablets or capsules, inhalation of powders, implantation, incorporationinto a matrix, or topical application of an encapsulated therapeuticfrom which the protein is gradually released over time.

The preparation of such sustained-release formulations are varied. Oneprocess includes mixing the protein with an organic solvent. Forexample, a powder formulation may be made by spraying a mixture of theprotein and an organic solvent into liquid nitrogen. Another processinvolves mixing the protein with a solution of abioerodible/biodegradable polymer in an organic solvent, resulting inthe formation of microparticles which contain the protein and thepolymer by coagulation of the mixture. In yet another process, proteins,powdered formulations, or microparticles can be mixed with an organicsolvent to produce a liquid or gel which may be injected into a patientor applied topically. Unfortunately, drawbacks to using organic solventsare their tendency to cause protein denaturation.

Additives have been used to stabilize proteins in the presence of adenaturing organic solvent. These additives include surfactants (seeU.S. Pat. No. 5,096,885), amino acids (see U.S. Pat. No. 4,297,344),polyols (see U.S. Pat. No. 5,589,167), natural polymers (see WO8903671), synthetic polymers (see Pharm. Res. 8:285 291, 1991), andmetals (see U.S. Pat. No. 6,191,107 B1), each of which is incorporatedherein by reference.

To date, no protein-based therapeutic agent (i.e. cytokines and growthfactors) is available that effectively recreates or mimics the complexcombination and physiologic levels of physiologically relevant cytokinesand growth factors found naturally in the body in healthy and disease orinjury states. Every protein-based therapeutic currently availableadministers a dose many, many times higher than the levels that thecytokines or growth factors are normally found in the body. In addition,no one has yet been able to administer these physiologically relevantcytokines and growth factors at physiological levels. Further, no onehas yet been able to administer these physiologically relevant cytokinesand growth factors at physiological levels in a sustained-releaseformulation. Therefore, Applicants present herewith for the first timethe instant invention whose object is to satisfy the unmet medical needof providing physiologically relevant growth factors and cytokines atphysiologic levels (CFS compositions), and in some instances, deliveringthose physiologically relevant growth factors and cytokines atphysiologic levels using a sustained-release formulation (SR-CFScompositions).

BRIEF SUMMARY OF THE INVENTION

It is an object of the instant invention to provide novel cellularfactor-containing solution (CFS) compositions that recreate the complexand unique combination and physiologic levels of such cytokines andgrowth factors found in biological niches. It is further an object ofthe instant invention to provide novel sustained-release cellularfactor-containing solution (SR-CFS) compositions that contain thecomplex and unique combination and physiologic levels of the cytokinesand growth factors found naturally in biological niches. Because thecellular factors are present in levels comparable to physiologicallevels found in the body, they are optimal for use in therapeuticapplications which require intervention to support, initiate, replace,accelerate or otherwise influence biochemical and biological processesinvolved in the treatment and/or healing of disease and/or injury. Inthe case of the SR-CFS compositions, the cellular factors are releasedslowly over time to provide a continual, consistent physiologic level ofsuch factors to optimize healing and/or recovery.

In addition to the novel CFS compositions described herein, it is alsoan object of the invention to provide methods for making such novel CFScompositions, including pooling cell-derived compositions (i.e.pooled-ECS and pooled-ACCS compositions), and SR-CFS compositions, aswell as therapeutic uses thereof.

The pooled cell-derived compositions possess several importantproperties and characteristics including decreased variability in thelevels of physiologically relevant cellular factors necessary fortherapeutic effect as compared to non-pooled compositions. The cellularfactors are present in levels comparable to physiological levels foundin the body and are thus optimal for use in therapeutic applicationswhich require intervention to support, initiate, replace, accelerate orotherwise influence biochemical and biological processes involved in thetreatment and/or healing of disease and/or injury. The novel methodsdescribed herein of pooling cell-derived compositions to decreasevariability has the effect of optimizing levels of the secreted factorssuch that their full therapeutic potential can be achieved in everypool. In addition to the therapeutic value of such pooled compositions,the method of pooling samples to decrease non-pooledcomposition-to-composition variability has the significant commercialadvantages of increasing production yields by minimizing non-pooledcomposition rejection for failure to meet product specifications and,consequently, decreasing production costs and increasing revenues.

Accordingly, a first aspect of the invention is an extraembryoniccell-derived cellular cytokine solution composition comprisingphysiologic concentrations of a) at least one factor selected from VEGF,TGFβ2, Angiogenin and PDGF; and b) at least one MMP inhibitor. Inanother embodiment the composition of aspect one comprises physiologicconcentrations of a) at least two factors selected from VEGF, TGFβ2,Angiogenin and PDGF; and b) at least one MMP inhibitor. In anotherembodiment the composition of aspect one comprises physiologicconcentrations of a) at least three factors selected from VEGF, TGFβ2,Angiogenin and PDGF; and b) at least one MMP inhibitor. In a specificembodiment the MMP inhibitor is selected from TIMP-1 and TIMP-2. In yetanother embodiment the composition of aspect one comprises physiologicconcentrations of VEGF, TGFβ2, Angiogenin, PDGF and TIMP-1. In stillanother embodiment the composition of aspect one comprises physiologicconcentrations of VEGF, TGFβ2, Angiogenin, PDGF and TIMP-2. In aspecific embodiment the composition of aspect one comprises physiologicconcentrations of VEGF, TGFβ2, Angiogenin, PDGF, TIMP-1 and TIMP-2.

Aspect two of the invention is one wherein the extraembryoniccell-derived cellular cytokine solution is amnion-derived cellularcytokine solution. In a specific embodiment of aspect two theamnion-derived cellular cytokine solution comprise physiologicconcentrations of VEGF, TGFβ2, Angiogenin, PDGF, TIMP-1 and TIMP-2.

Aspect three of the invention is a sustained-release compositioncomprising the extraembryonic cell-derived cellular cytokine solution ofaspect one or the amnion-derived cellular cytokine solution of aspecttwo.

Aspect four of the invention is a method of making an amnion-derivedcellular cytokine solution comprising a) isolating amnion epithelialcells from the amnion of a placenta, b) selecting AMP cells from theamnion epithelial cells, c) culturing the AMP cells until they reachconfluence, d) changing the culture medium, e) culturing the cells inthe medium, and f) collecting the culture medium of step (e) to obtainamnion-derived cellular cytokine solution. In a particular embodiment ofaspect four, step (f) is repeated a plurality of times and theamnion-derived cellular cytokine solution obtained in each step (f) iscombined to create a pooled amnion-derived cellular cytokine solution.

Aspect five of the invention is the amnion-derived cellular cytokinesolution made by the method of aspect four. In one embodiment, theamnion-derived cellular cytokine solution of aspect five is one whereinthe solution comprises the factors VEGF, TGFβ2, Angiogenin, PDGF, TIMP-1and/or TIMP-2 at physiological concentrations. In certain embodiments ofthe compositions of the invention, the physiologic concentration is˜5.0-16 ng/mL for VEGF, ˜3.5-4.5 ng/mL for Angiogenin, ˜100-165 pg/mLfor PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μg mL for TIMP-1 and ˜1.04μg/mL for TIMP-2.

Aspect six of the invention is a physiologic cytokine solutioncomposition comprising a therapeutic component consisting essentially ofphysiologic concentrations of: a) one or more factors selected fromVEGF, TGFβ2, Angiogenin and PDGF; b) at least MMP inhibitor; and c) acarrier, wherein the physiologic concentration is ˜5.0-16 ng/mL forVEGF, ˜3.5-4.5 ng/mL for Angiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7ng/mL for TGFβ2, and wherein the carrier is normal saline, PBS, lactatedRinger's solution or cell culture medium. In one embodiment of aspectsix the MMP inhibitor is TIMP-1 and/or TIMP-2 and the physiologicconcentration is ˜0.68 μg mL for TIMP-1 and ˜1.04μg/mL for TIMP-2.Another embodiment of aspect six comprises a therapeutic componentconsisting essentially of physiologic concentrations of: a) VEGF, TGFβ2,Angiogenin and PDGF; b) TIMP-1 and/or TIMP-2; and c) a carrier; whereinthe physiologic concentration is ˜5.0-16 ng/mL for VEGF, ˜3.5-4.5 ng/mLfor Angiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68μg mL for TIMP-1 and ˜1.04μg/mL for TIMP-2, and wherein the carrier isnormal saline, PBS, lactated Ringer's solution or cell culture medium.

Aspect seven of the invention is a physiologic cytokine solutioncomposition comprising a therapeutic component consisting essentially ofa composition selected from the group consisting of: Composition A: VEGFand TIMP-1; Composition B: VEGF, Angiogenin and TIMP-1; Composition C:VEGF, Angiogenin, PDGF-BB and TIMP-1; Composition D: VEGF, Angiogenin,PDGF-BB, TGFβ2 and TIMP-1; Composition E: VEGF and TIMP-2; CompositionF: VEGF, Angiogenin and TIMP-2; Composition G: VEGF, Angiogenin, PDGF-BBand TIMP-2; Composition H: VEGF, Angiogenin, PDGF-BB, TGFβ2 and TIMP-2;Composition I: VEGF, TIMP-1 and TIMP-2; Composition J: VEGF, Angiogenin,TIMP-1 and TIMP-2; Composition K: VEGF, Angiogenin, PDGF-BB, TIMP-1 andTIMP-2; Composition L: VEGF, Angiogenin, PDGF-BB, TGFβ2, TIMP-1 andTIMP-2; Composition M: Angiogenin and TIMP-1; Composition N: Angiogenin,PDGF-BB and TIMP-1; Composition O: Angiogenin, PDGF-BB, TGFβ2 andTIMP-1; Composition P: Angiogenin and TIMP-2; Composition Q: Angiogenin,PDGF-BB and TIMP-2; Composition R: Angiogenin, PDGF-BB, TGFβ2 andTIMP-2; Composition S: Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2;Composition T: PDGF-BB and TIMP-1; Composition U: PDGF-BB, TGFβ2 andTIMP-1; Composition V: PDGF-BB and TIMP-2; Composition W: PDGF-BB, TGFβ2and TIMP-2; Composition X: PDGF-BB, TIMP-1 and TIMP-2; and CompositionY: PDGF-BB, TGFβ2, TIMP-1 and TIMP-2; and a carrier, wherein VEGF,Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2 are at ˜5-16 ng/mL forVEGF, ˜3.5-4.5 ng/mL for Angiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7ng/mL for TGFβ2, ˜0.68 μg mL for TIMP-1 and ˜1.04μg/mL for TIMP-2, andwherein the carrier is normal saline, PBS, lactated Ringer's solution orcell culture medium.

Aspect eight of the invention is a sustained-release compositioncomprising the composition of aspects 6 and 7.

Aspect nine of the invention is the sustained-release composition ofaspect eight further comprising an agent capable of effectingsustained-release of the extraembryonic cell-derived cellular cytokinesolution, wherein the agent is selected from polymers, blended polymers,hyaluronic acid particles, microencapsulation materials ornanoparticles.

Aspect ten of the invention is a method of making a sustained-releasecomposition comprising the steps of combining a cellularfactor-containing solution composition with an agent capable ofeffecting sustained-release of the cellular factor-containing solutioncomposition.

Aspect 11 of the invention is the sustained-release composition made bythe method of aspect 10.

DEFINITIONS

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state.

As used herein, the term “protein marker” means any protein moleculecharacteristic of the plasma membrane of a cell or in some cases of aspecific cell type.

As used herein, “enriched” means to selectively concentrate or toincrease the amount of one or more materials by elimination of theunwanted materials or selection and separation of desirable materialsfrom a mixture (i.e. separate cells with specific cell markers from aheterogeneous cell population in which not all cells in the populationexpress the marker).

As used herein, the term “substantially purified” means a population ofcells substantially homogeneous for a particular marker or combinationof markers. By substantially homogeneous is meant at least 90%, andpreferably 95% homogeneous for a particular marker or combination ofmarkers.

The term “placenta” as used herein means both preterm and term placenta.

As used herein, the term “totipotent cells” shall have the followingmeaning. In mammals, totipotent cells have the potential to become anycell type in the adult body; any cell type(s) of the extraembryonicmembranes (e.g., placenta). Totipotent cells are the fertilized egg andapproximately the first 4 cells produced by its cleavage.

As used herein, the term “pluripotent stem cells” shall have thefollowing meaning. Pluripotent stem cells are true stem cells with thepotential to make any differentiated cell in the body, but cannotcontribute to making the components of the extraembryonic membraneswhich are derived from the trophoblast. The amnion develops from theepiblast, not the trophoblast. Three types of pluripotent stem cellshave been confirmed to date: Embryonic Stem (ES) Cells (may also betotipotent in primates), Embryonic Germ (EG) Cells, and EmbryonicCarcinoma (EC) Cells. These EC cells can be isolated fromteratocarcinomas, a tumor that occasionally occurs in the gonad of afetus. Unlike the other two, they are usually aneuploid.

As used herein, the term “multipotent stem cells” are true stem cellsbut can only differentiate into a limited number of types. For example,the bone marrow contains multipotent stem cells that give rise to allthe cells of the blood but may not be able to differentiate into othercells types.

As used herein, the term “extraembryonic tissue” means tissue locatedoutside the embryonic body which is involved with the embryo'sprotection, nutrition, waste removal, etc. Extraembryonic tissue isdiscarded at birth. Extraembryonic tissue includes but is not limited tothe amnion, chorion (trophoblast and extraembryonic mesoderm includingumbilical cord and vessels), yolk sac, allantois and amniotic fluid(including all components contained therein). Extraembryonic tissue andcells derived therefrom have the same genotype as the developing embryo.

As used herein, the term “extraembryonic cytokine secreting cells” or“ECS cells” means a population of cells derived from the extraembryonictissue which have the characteristics of secreting a unique combinationof physiologically relevant cytokines in a physiologically relevanttemporal manner into the extracellular space or into surrounding culturemedia and which have not been cultured in the presence of anyanimal—derived products, making them and cell products derived from themsuitable for human clinical use. In one embodiment, the ECS cellssecrete at least one cytokine selected from VEGF, Angiogenin, PDGF andTGFβ2 and at least one MMP inhibitor. In a specific embodiment, the MMPinhibitor is selected from TIMP-1 and TIMP-2. In another embodiment, theECS cells secrete more than one cytokine selected from VEGF, Angiogenin,PDGF and TGFβ2 and more than one MMP inhibitor. In a specificembodiment, the MMP inhibitor is selected from TIMP-1 and TIMP-2. In apreferred embodiment, the ECS cells secrete the cytokines VEGF,Angiogenin, PDGF and TGFβ2 and the MMP inhibitors TIMP-1 and/or TIMP-2.The physiological range of the cytokine or cytokines in the uniquecombination is as follows: ˜5-16 ng/mL for VEGF, ˜3.5-4.5 ng/mL forAngiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μgmL for TIMP-1 and ˜1.04 μg/mL for TIMP-2. The ECS cells may optionallyexpress Thymosin β4. ECS cells may be selected from populations of cellsand compositions described in this application and in US2003/0235563,US2004/0161419, US2005/0124003, U.S. Provisional Application Nos.60/666,949, 60/699,257, 60/742,067, 60/813,759, U.S. application Ser.No. 11/333,849, U.S. application Ser. No. 11/392,892, PCTUS06/011392,US2006/0078993, PCT/US00/40052, U.S. Pat. No. 7,045,148, US2004/0048372,and US2003/0032179, the contents of which are incorporated herein byreference in their entirety.

As used herein, the term “amnion-derived multipotent progenitor cell” or“AMP cell” means a specific population of ECS cells that are epithelialcells derived from the amnion. In addition to the characteristicsdescribed above for ECS cells, AMP cells have the followingcharacteristics. They have not been cultured in the presence of anyanimal—derived products, making them and cell products derived from themsuitable for human clinical use. They grow without feeder layers, do notexpress the protein telomerase and are non-tumorigenic. AMP cells do notexpress the hematopoietic stem cell marker CD34 protein. The absence ofCD34 positive cells in this population indicates the isolates are notcontaminated with hematopoietic stem cells such as umbilical cord bloodor embryonic fibroblasts. Virtually 100% of the cells react withantibodies to low molecular weight cytokeratins, confirming theirepithelial nature. Freshly isolated AMP cells will not react withantibodies to the stem/progenitor cell markers c-kit (CD117) and Thy-1(CD90). Several procedures used to obtain cells from full term orpre-term placenta are known in the art (see, for example, US2004/0110287; Anker et al., 2005, Stem Cells 22:1338-1345; Ramkumar etal., 1995, Am. J. Ob. Gyn. 172:493-500). However, the methods usedherein provide improved compositions and populations of cells. AMP cellshave previously been described as “amnion-derived cells” (see U.S.Provisional Application Nos. 60/666,949, 60/699,257, 60/742,067, U.S.Provisional Application Nos. 60/813,759, U.S. application Ser. No.11/333,849, U.S. application Ser. No. 11/392,892, and PCTUS06/011392,each of which is incorporated herein in its entirety).

By the term “animal-free” when referring to certain compositions, growthconditions, culture media, etc. described herein, is meant that noanimal-derived materials, such as animal-derived serum, other thanclinical grade human materials, such as recombinantly produced humanproteins, are used in the preparation, growth, culturing, expansion,storage or formulation of the certain composition or process.

By the term “serum-free” when referring to certain compositions, growthconditions, culture media, etc. described herein, is meant that noanimal-derived serum (i.e. no non-human) is used in the preparation,growth, culturing, expansion, storage or formulation of the certaincomposition or process.

By the term “expanded”, in reference to cell compositions, means thatthe cell population constitutes a significantly higher concentration ofcells than is obtained using previous methods. For example, the level ofcells per gram of amniotic tissue in expanded compositions of AMP cellsis at least 50 and up to 150 fold higher than the number of cells in theprimary culture after 5 passages, as compared to about a 20 foldincrease in such cells using previous methods. In another example, thelevel of cells per gram of amniotic tissue in expanded compositions ofAMP cells is at least 30 and up to 100 fold higher than the number ofcells in the primary culture after 3 passages. Accordingly, an“expanded” population has at least a 2 fold, and up to a 10 fold,improvement in cell numbers per gram of amniotic tissue over previousmethods. The term “expanded” is meant to cover only those situations inwhich a person has intervened to elevate the number of the cells.

As used herein, the term “passage” means a cell culture technique inwhich cells growing in culture that have attained confluence or areclose to confluence in a tissue culture vessel are removed from thevessel, diluted with fresh culture media (i.e. diluted 1:5) and placedinto a new tissue culture vessel to allow for their continued growth andviability. For example, cells isolated from the amnion are referred toas primary cells. Such cells are expanded in culture by being grown inthe growth medium described herein. When such primary cells aresubcultured, each round of subculturing is referred to as a passage. Asused herein, “primary culture” means the freshly isolated cellpopulation.

As used herein, “conditioned medium” is a medium in which a specificcell or population of cells has been cultured, and then removed. Whencells are cultured in a medium, they may secrete cellular factors thatcan provide support to or affect the behavior of other cells. Suchfactors include, but are not limited to hormones, cytokines,extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines,receptors, inhibitors and granules. The medium containing the cellularfactors is the conditioned medium. Examples of methods of preparingconditioned media are described in U.S. Pat. No. 6,372,494 which isincorporated by reference in its entirety herein. As used herein,conditioned medium also refers to components, such as proteins, that arerecovered and/or purified from conditioned medium or from ECS cells,including AMP cells.

As used herein, the term “cellular factor-containing solution” or “CFS”composition means a composition having physiologic concentrations of oneor more factors selected from VEGF, Angiogenin, PDGF and TGFβ2 and atleast one MMP inhibitor. Examples of suitable MMP inhibitors include butare not limited to TIMP-1 and TIMP-2. CFS compositions includeconditioned media derived from ECS cells, amnion-derived cellularcytokine solution compositions (see definition below), physiologiccytokine solution compositions (see definition below), and sustainedrelease formulations of such CFS compositions.

As used herein, the term “amnion-derived cellular cytokine solution” or“ACCS” means conditioned medium that has been derived from AMP cells orexpanded AMP cells. Amnion-derived cellular cytokine solution or ACCShas previously been referred to as “amnion-derived cytokine suspension”.

As used herein, the term “physiologic cytokine solution” or “PCS”composition means a composition which is not cell-derived and which hasphysiologic concentrations of one or more factors selected from VEGF,Angiogenin, PDGF and TGFβ2 and at least one MMP inhibitor. Examples ofsuitable MMP inhibitors include but are not limited to TIMP-1 andTIMP-2.

As used herein, the term “suspension” means a liquid containingdispersed components, i.e. cytokines. The dispersed components may befully solubilized, partially solubilized, suspended or otherwisedispersed in the liquid. Suitable liquids include, but are not limitedto, water, osmotic solutions such as salt and/or sugar solutions, cellculture media, and other aqueous or non-aqueous solutions.

The term “lysate” as used herein refers to the composition obtained whencells, for example, AMP cells, are lysed and optionally the cellulardebris (e.g., cellular membranes) is removed. This may be achieved bymechanical means, by freezing and thawing, by sonication, by use ofdetergents, such as EDTA, or by enzymatic digestion using, for example,hyaluronidase, dispase, proteases, and nucleases.

The term “physiologic” or “physiological level” as used herein means thelevel that a substance in a living system is found and that is relevantto the proper functioning of a biochemical and/or biological process.

As used herein, the term “pooled” means a plurality of compositions thathave been combined to create a new composition having more constant orconsistent characteristics as compared to the non-pooled compositions.For example, pooled ACCS has more constant or consistent characteristicscompared to non-pooled ACCS. Examples of pooled compositions include “SPpools” (more than one ACCS collection/one placenta), “MP1 pools” (oneACCS collection/placenta, multiple placentas), and “MP2 pools” (morethan one ACCS collection/placenta, multiple placentas).

As used herein, the term “substrate” means a defined coating on asurface that cells attach to, grown on, and/or migrate on. As usedherein, the term “matrix” means a substance that cells grow in or onthat may or may not be defined in its components. The matrix includesboth biological and non-biological substances. As used herein, the term“scaffold” means a three-dimensional (3D) structure (substrate and/ormatrix) that cells grow in or on. It may be composed of biologicalcomponents, synthetic components or a combination of both. Further, itmay be naturally constructed by cells or artificially constructed. Inaddition, the scaffold may contain components that have biologicalactivity under appropriate conditions.

The term “cell product” or “cell products” as used herein refers to anyand all substances made by and secreted from a cell, including but notlimited to, protein factors (i.e. growth factors, differentiationfactors, engraftment factors, cytokines, morphogens, proteases (i.e. topromote endogenous cell delamination, protease inhibitors),extracellular matrix components (i.e. fibronectin, etc.).

The term “therapeutically effective amount” means that amount of atherapeutic agent necessary to achieve a desired physiological effect(i.e. accelerate wound healing).

As used herein, the term “pharmaceutically acceptable” means that thecomponents, in addition to the therapeutic agent, comprising theformulation, are suitable for administration to the patient beingtreated in accordance with the present invention.

As used herein, the term “therapeutic component” means a component ofthe composition which exerts a therapeutic benefit when the compositionis administered to a subject.

As used herein, the term “therapeutic protein” includes a wide range ofbiologically active proteins including, but not limited to, growthfactors, enzymes, hormones, cytokines, inhibitors of cytokines, bloodclotting factors, peptide growth and differentiation factors.

As used herein, the term “tissue” refers to an aggregation of similarlyspecialized cells united in the performance of a particular function.

As used herein, the terms “a” or “an” means one or more; at least one.

As used herein, the term “adjunctive” means jointly, together with, inaddition to, in conjunction with, and the like.

As used herein, the term “co-administer” can include simultaneous orsequential administration of two or more agents.

As used herein, the term “agent” means an active agent or an inactiveagent. By the term “active agent” is meant an agent that is capable ofhaving a physiological effect when administered to a subject.Non-limiting examples of active agents include growth factors,cytokines, antibiotics, cells, conditioned media from cells, etc. By theterm “inactive agent” is meant an agent that does not have aphysiological effect when administered. Such agents may alternatively becalled “pharmaceutically acceptable excipients”. Non-limiting examplesinclude time release capsules and the like.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articulare, subcapsular, subarachnoid, intraspinal, epidural,intracerebral and intrasternal injection or infusion.

The terms “sustained-release”, “extended-release”, “time-release”,“controlled-release”, or “continuous-release” as used herein means anagent, typically a therapeutic agent or drug, that is formulated todissolve slowly and be released over time.

The terms “bioerodable” or “bioerosion” as used herein mean acombination of physical (i.e. dissolution) and chemical (i.e. chemicalbond cleavage) processes that result in the breakdown of a substance.

The term “biodegradable” or “biodegradation” as used herein means abiological agent (i.e. an enzyme, microbe or cell) is responsible forthe breakdown of a substance.

The terms “bioresporbable” or “bioabsorptable” as used herein mean theremoval of a breakdown product by cellular activity (i.e. phagocytosis).

As used herein, the term “nanoparticle” means particles of less than 100nm in diameter that exhibit new or enhanced size-dependent propertiescompared with larger particles of the same material.

“Treatment,” “treat,” or “treating,” as used herein covers any treatmentof a disease or condition of a mammal, particularly a human, andincludes: (a) preventing the disease or condition from occurring in asubject which may be predisposed to the disease or condition but has notyet been diagnosed as having it; (b) inhibiting the disease orcondition, i.e., arresting its development; (c) relieving and orameliorating the disease or condition, i.e., causing regression of thedisease or condition; or (d) curing the disease or condition, i.e.,stopping its development or progression. The population of subjectstreated by the methods of the invention includes subjects suffering fromthe undesirable condition or disease, as well as subjects at risk fordevelopment of the condition or disease.

As used herein, a “wound” is any disruption, from whatever cause, ofnormal anatomy (internal and/or external anatomy) including but notlimited to traumatic injuries such as mechanical (i.e. contusion,penetrating), thermal, chemical, electrical, radiation, concussive andincisional injuries; elective injuries such as operative surgery andresultant incisional hernias, fistulas, etc.; acute wounds, chronicwounds, infected wounds, and sterile wounds, as well as woundsassociated with disease states (i.e. ulcers caused by diabeticneuropathy or ulcers of the gastrointestinal or genitourinary tract). Awound is dynamic and the process of healing is a continuum requiring aseries of integrated and interrelated cellular processes that begin atthe time of wounding and proceed beyond initial wound closure througharrival at a stable scar. These cellular processes are mediated ormodulated by humoral substances including but not limited to cytokines,lymphokines, growth factors, and hormones. In accordance with thesubject invention, “wound healing” refers to improving, by some form ofintervention, the natural cellular processes and humoral substances oftissue repair such that healing is faster, and/or the resulting healedarea has less scaring and/or the wounded area possesses tissue strengththat is closer to that of uninjured tissue and/or the wounded tissueattains some degree of functional recovery.

As used herein the term “standard animal model for wound healing” refersto any art-accepted animal model for wound healing in which thecompositions of the invention exhibit efficacy as measured byaccelerated wound healing. Non-limiting examples of suitable models aredescribed in Hayward P G, Robson M C: Animal models of woundcontraction. In Barbul A, et al: Clinical and Experimental Approaches toDermal and Epidermal Repair: Normal and Chronic Wounds. John Wiley &Sons, New York, 1990; DelBecarro, et al: The use of specific thromboxaneinhibitors to preserve the dermal microcirculation after burning.Surgery 87: 137-141, 1980; Robson, et al: Increasing dermal perfusionafter burning by decreasing thromboxane production. J Trauma 20:722-725, 1980; Polo, et al: An in vivo model of human proliferativescar. J Surg Res 74: 187-195, 1998). Skilled artisans are aware of othersuitable models.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized CellsAnd Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Obtaining and Culturing of Cells

ECS cells—Various methods for isolating cells from the extraembryonictissue, which may then be used to produce the ECS cells of the instantinvention are described in the art (see, for example, US2003/0235563,US2004/0161419, US2005/0124003, U.S. Provisional Application Nos.60/666,949, 60/699,257, 60/742,067, 60/813,759, U.S. application Ser.No. 11/333,849, U.S. application Ser. No. 11/392,892, PCTUS06/011392,US2006/0078993, PCT/US00/40052, U.S. Pat. No. 7,045,148, US2004/0048372,and US2003/0032179).

Identifying ECS cells—Once extraembryonic tissue is isolated, it isnecessary to identify which cells in the tissue have the characteristicsassociated with ECS cells (see definition above). For example, cells areassayed for their ability to secrete a unique combination of cytokinesinto the extracellular space or into surrounding culture medium.Suitable cells are those in which the cytokine or cytokines occurs inthe physiological range of ˜5.0-16 ng/mL for VEGF, ˜3.5-4.5 ng/mL forAngiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μgmL for TIMP-1 and ˜1.04μg/mL for TIMP-2. Suitable cells may optionallysecrete Thymosin (34.

AMP cells—In a particular embodiment, AMP cell compositions are preparedusing the steps of a) recovery of the amnion from the placenta, b)dissociation of the amnion epithelial cells from the amniotic membrane,c) isolating AMP cells from the amnion epithelial cells, d) culturing ofthe AMP cells in a basal medium with the addition of a naturally derivedor recombinantly produced human protein; and optionally d) furtherproliferation of the cells using additional additives and/or growthfactors. Details are contained in US Publication No. 2006-0222634-A1,which is incorporated herein by reference.

AMP cells are cultured as follows: The AMP cells are cultured in a basalmedium. Such medium includes, but is not limited to, Epilife (CascadeBiologicals), Opti-pro, VP-SFM, IMDM, Advanced DMEM, K/0 DMEM, 293 SFMII (all made by Gibco; Invitrogen), HPGM, Pro 2935-CDM, Pro 293A-CDM,UltraMDCK, UltraCulture (all made by Cambrex), Stemline I and StemlineII (both made by Sigma-Aldrich), DMEM, DMEM/F-12, Ham's F12, M199, andother comparable basal media. Such media may optionally contain clinicalgrade human protein or be supplemented with human clinical gradeprotein. As used herein a “human protein” is one that is producednaturally or one that is produced using recombinant technology. Incertain specific embodiments, “human protein” also is meant to include ahuman fluid or derivative or preparation thereof, such as human serum oramniotic fluid, which contains human protein.

In a most preferred embodiment, the cells are cultured using a systemthat is free of animal products to avoid xeno-contamination. In thisembodiment, the culture medium is Stemline I or II, Opti-pro, IMDM orDMEM, and is optionally supplemented with clinical grade human albuminadded up to concentrations of 10%. In preferred embodiments, the mediumis serum-free in addition to being animal-free.

Optionally, other factors are used. In one embodiment, epidermal growthfactor (EGF) at a concentration of between 0-1 μg/mL is used. In apreferred embodiment, the EGF concentration is around 10 ng/mL.Alternative growth factors which may be used include, but are notlimited to, TGFα or TGFβ2 (5 ng/mL; range 0.1-100 ng/mL), activin A,cholera toxin (preferably at a level of about 0.1 μg/mL; range 0-10μg/mL), transferrin (5 μg/mL; range 0.1-100 μg/mL), fibroblast growthfactors (bFGF 40 ng/mL (range 0-200 ng/mL), aFGF, FGF-4, FGF-8; (all inrange 0-200 ng/mL), bone morphogenic proteins (i.e. BMP-4) or othergrowth factors known to enhance cell proliferation. All supplements areclinical grade.

Generation of Cellular Factor-Containing Solution Conditioned Medium

Generation of ECS conditioned medium—is obtained as described below forACCS, except that ECS cells are used.

Generation of ACCS—The AMP cells of the invention can be used togenerate ACCS. In one embodiment, the AMP cells are isolated asdescribed herein and 1×10⁶ cells/mL are seeded into T75 flaskscontaining between 5-30 mL culture medium, preferably between 10-25 mLculture medium, and most preferably about 10 mL culture medium. Thecells are cultured until confluent, the medium is changed and in oneembodiment the ACCS is collected 1 day post-confluence. In anotherembodiment the medium is changed and ACCS is collected 2 dayspost-confluence. In another embodiment the medium is changed and ACCS iscollected 4 days post-confluence. In another embodiment the medium ischanged and ACCS is collected 5 days post-confluence. In a preferredembodiment the medium is changed and ACCS is collected 3 dayspost-confluence. In another preferred embodiment the medium is changedand ACCS is collected 3, 4, 5, 6 or more days post-confluence. Skilledartisans will recognize that other embodiments for collecting ACCS fromAMP cell cultures, such as using other tissue culture vessels, includingbut not limited to cell factories, flasks, hollow fibers, or suspensionculture apparatus, or collecting ACCS from sub-confluent and/or activelyproliferating cultures, are also contemplated by the methods of theinvention.

It is also contemplated by the instant invention that the ACCS becryopreserved following collection. It is also contemplated by theinvention that ACCS be lyophilized following collection. It is alsocontemplated by the invention that ACCS be formulated forsustained-release following collection. It is also contemplated thatACCS production be scaled up for generation of sufficient product forclinical testing and for commercialization. Skilled artisans arefamiliar with cryopreservation lyophilization, and sustained-releaseformulation methodologies.

It is also contemplated by the invention that CFS compositions such asACCS and pooled ACCS, be diluted with appropriate diluent prior to use.Appropriate diluents include, without limitation, normal saline, PBS,lactated Ringer's solution, cell culture media, conditioned cell culturemedia, water, and the like. Such dilutions may be 1:2, 1:3, 1:4, 1:5,1:10, 1:100, etc. In addition, dilutions may be less than 1:2 (i.e. 1:1,1:0.5, etc.) The appropriate dilution required will be dependent uponthe intended use and therefore will need to be empirically determined byskilled artisans.

The CFS compositions of the invention, including ACCS, pooled ACCS, PCS,etc., are characterized by assaying for physiologically relevantcytokines in the physiologically relevant range of ˜5-16 ng/mL for VEGF,˜3.5-4.5 ng/mL for Angiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mLfor TGFβ2, ˜0.68 μg mL for TIMP-1 and ˜1.04 μg/mL for TIMP-2. ACCS andpooled ACCS are optionally assayed for the presence of Thymosin β4.

Generation of Physiologic Cytokine Solution (PCS) Compositions

A non-cellular derived form of ACCS (referred to herein as PhysiologicCytokine Solution (“PCS”) composition is generated by combiningphysiological levels of one or more of VEGF, Angiogenin, PDGF, TGFβ2,and one or more MMP inhibitor (i.e. TIMP-1 and/or TIMP-2) in a carrier.Optionally, the PCS contains Thymosinβ4. The physiological levels forthese cytokines are the same as those found in ACCS. Suitable carriersinclude normal saline, PBS, lactated Ringer's solution, cell culturemedium, conditioned cell culture media, water, etc. Such compositionsare suitable for cryopreservation, lyophilization, sustained-releaseformulation, scale-up, and the like. It is contemplated by the presentinvention that PCS may be produced such that it contains moreconcentrated levels of the factors than those found in ACCS and that itmay be subsequently diluted with appropriate diluent prior to use.Appropriate diluents include, without limitation, normal saline, PBS,lactated Ringer's solution, cell culture media, conditioned cell culturemedia, water, and the like. Such dilutions may be 1:2, 1:3, 1:4, 1:5,1:10, 1:100, etc. Such dilutions may also be less than 1:2 (i.e. 1:1,1:0.5, etc.). The appropriate dilution required will be dependent uponthe intended use and therefore will need to be empirically determined bythe skilled artisan.

The compositions of the invention can be prepared in a variety of waysdepending on their intended use. For example, a composition may be aliquid comprising an agent of the invention, i.e. ACCS, pooled ACCS, andPCS. A liquid composition also includes a gel. The liquid compositionmay be aqueous or in the form of an ointment, salve, cream, or the like.The liquid composition may also be formulated in such a way as to besuitable for a spray or aerosol application.

A useful aqueous suspension may contain one or more polymers assuspending agents. Useful polymers include water-soluble polymers suchas cellulosic polymers and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. An aqueous suspension orsolution/suspension of the present invention is preferably viscous ormuco-adhesive, or even more preferably, both viscous and muco-adhesive.

Sustained-Release Compositions

The CFS compositions, including but not limited to ACCS, pooled ACCS andPCS, maybe formulated as sustained-release CFS compositions (referred toherein as “SR-CFS”). Skilled artisans are familiar with methodologies tocreate sustained-release compositions of therapeutic agents, includingprotein-based therapeutic agents such as ACCS, pooled ACCS or PCS.

SR-CFS, including but not limited to SR-ACCS and SR-PCS, may be made byany of the methods described herein. For example, multivesicularliposome formulation technology is useful for the sustained-release ofprotein and peptide therapeutics. Qui, J., et al, (ACTA Pharmacol Sin,2005, 26(11):1395-401) describe this methodology for the formulation ofsustained-release interferon alpha-2b. Vyas, S. P., et al, (Drug Dev IndPharm, 2006, 32(6):699-707) describe encapsulating pegylated interferonalpha in multivesicular liposomes. ACCS (including pooled ACCS) and PCSare suitable for use in multivesicular liposome sustained-releaseformulation.

Nanoparticle technology is also useful for creating SR-CFS. For example,Packhaeuser, C. B., et al, (J Control Release, 2007, 123(2):131-40)describe biodegradable parenteral depot systems based on insulin loadeddialkylaminoalkyl-amine-poly(vinyl alcohol)-g-poly(lactide-co-glycolide)nanoparticules and conclude that nanoparticle-based depots are suitablecandidates for the design of controlled-release devices for bioactivemacromolecules (i.e. proteins). Dailey, L. A., et al, (Pharm Res 2003,20(12):2011-20) describe surfactant-free, biodegradable nanoparticlesfor aerosol therapy which is based on the branched polymersDEAPA-PVAL-g-PLGA and conclude that DEAPA-PVAL-g-PLGA are versatile drugdelivery systems. ACCS (including pooled ACCS) and PCS are suitable foruse in nanoparticle-based sustained-release formulations.

Polymer-based sustained-release formulations are also very useful. Chan,Y. P., et al, (Expert Opin Drug Deliv, 2007, 4(4):441-51) provide areview of the Medusa system (Flamel Technologies), which is used forsustained-release of protein and peptide therapies. Thus far, the Medusasystem has been applied to subcutaneous injection of IL-2 andIFN-alpha(2b), in animal models (rats, dogs, monkeys), and in clinicaltrials in renal cancer (IL-2) and hepatitis C (IFN-alpha(2b)) patients.Chavanpatil, M. D., et al, (Pharm Res, 2007, 24(4):803-10) describesurfactant-polymer nanoparticles as a novel platform for sustained andenhanced cellular delivery of water-soluble molecules. Takeuchi, H., etal, (Adv Drug Deliv Res, 2001, 47(1):39-54) describe mucoadhesivenanoparticulate systems for peptide drug delivery, including liposomesand polymeric nanoparticles. Wong, H. L., et al, (Pharm Res, 2006,23(7):1574-85) describe a new polymer-lipid hybrid system which has beenshown to increase cytotoxicity of doxorubicin againstmultidrug-resistant breast cancer cells. CFS compositions, including butnot limited to ACCS (including pooled ACCS) and PCS are suitable for usein the aforementioned sustained-release formulation methodologies.

In addition, other sustained-release methodologies familiar to skilledartisans, while not specifically described herein, are also suitable foruse with the CFS compositions.

Pharmaceutical Compositions of CFS Compositions Including, But notLimited to, ACCS, Pooled ACCS, PCS, SR-ACCS (including pooled ACCS), andSR-PCS

The present invention provides pharmaceutical compositions of CFScompositions and a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the composition isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, and still othersare familiar to skilled artisans.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Treatment Kits Comprising CFS Compositions Including, but not Limitedto, ACCS, Pooled ACCS, PCS, SR-ACCS (including Pooled ACCS), and SR-PCS

The invention also provides for an article of manufacture comprisingpackaging material and a pharmaceutical composition of the inventioncontained within the packaging material, wherein the pharmaceuticalcomposition comprises CFS compositions. The packaging material comprisesa label or package insert which indicates that the CFS compositionscontained therein can be used for therapeutic applications such as, forexample, wound healing.

Formulation, Dosage and Administration of CFS Compositions Including,but not Limited to, ACCS, Pooled ACCS, PCS, SR-ACCS (including PooledACCS), and SR-PCS

Compositions comprising CFS compositions may be administered to asubject to provide various cellular or tissue functions, for example, toaccelerate wound healing. As used herein “subject” may mean either ahuman or non-human animal.

Such compositions may be formulated in any conventional manner using oneor more physiologically acceptable carriers optionally comprisingexcipients and auxiliaries. Proper formulation is dependent upon theroute of administration chosen. The compositions may be packaged withwritten instructions for their therapeutic use. The compositions mayalso be administered to the recipient in one or more physiologicallyacceptable carriers. Carriers for CFS compositions may include but arenot limited to solutions of normal saline, phosphate buffered saline(PBS), lactated Ringer's solution containing a mixture of salts inphysiologic concentrations, or cell culture medium.

In addition, one of skill in the art may readily determine theappropriate dose of the CFS compositions for a particular purpose. Apreferred dose is in the range of about 0.1-to-1000 micrograms persquare centimeter of applied area. Other preferred dose ranges are1.0-to-50.0 micrograms/applied area. In a particularly preferredembodiment, it has been found that relatively small amounts of the CFScompositions are therapeutically useful. One exemplification of suchtherapeutic utility is the ability for ACCS (including pooled ACCS) toaccelerate wound healing (for details see U.S. Publication No.2006/0222634 and U.S. Publication No. 2007/1231297, each incorporatedherein by reference). One of skill in the art will also recognize thatthe number of doses to be administered needs also to be empiricallydetermined based on, for example, severity and type of disease, disorderor injury being treated. For example, in a preferred embodiment, onedose is sufficient to have a therapeutic effect (i.e. accelerate woundhealing). Other preferred embodiments contemplate, 2, 3, 4, or moredoses for therapeutic effect.

The skilled artisan will recognize that a preferred dose is one whichproduces a therapeutic effect (a therapeutically effective amount) suchas accelerating wound healing, in a patient in need thereof. Of course,proper doses of the CFS compositions will require empiricaldetermination at time of use based on several variables including butnot limited to the severity and type of injury, disorder or conditionbeing treated; patient age, weight, sex, health; other medications andtreatments being administered to the patient; and the like. One of skillin the art will also recognize that number of doses (dosing regimen) tobe administered needs also to be empirically determined based on, forexample, severity and type of injury, disorder or condition beingtreated. In addition, one of skill in the art recognizes that thefrequency of dosing needs to be empirically determined based on severityand type of injury, disorder or condition being treated. In certainembodiments, one dose is administered every day for a given number ofdays (i.e. once a day for 7 days, etc.). In other embodiments, multipledoses may be administered in one day (every 4 hours, etc.). Multipledoses per day for multiple days is also contemplated by the invention.

In further embodiments of the present invention, at least one additionalagent may be combined with the CFS compositions. Such agents may actsynergistically with the CFS compositions of the invention to enhancethe therapeutic effect. Such agents include but are not limited togrowth factors, cytokines, chemokines, antibodies, inhibitors,antibiotics, immunosuppressive agents, steroids, anti-fungals,anti-virals or other cell types (i.e. stem cells or stem-like cells, forexample AMP cells). Inactive agents include carriers, diluents,stabilizers, gelling agents, delivery vehicles, ECMs (natural andsynthetic), scaffolds, and the like. When the CFS compositions areadministered conjointly with other pharmaceutically active agents, evenless of the CFS compositions may be needed to be therapeuticallyeffective.

CFS compositions can be administered by injection into a target site ofa subject, preferably via a delivery device, such as a tube, e.g.,catheter. In a preferred embodiment, the tube additionally contains aneedle, e.g., a syringe, through which the CFS compositions can beintroduced into the subject at a desired location. Specific,non-limiting examples of administering the CFS compositions to subjectsmay also include administration by subcutaneous injection, intramuscularinjection, intravenous injection, intraarterial injection, intracardiacinjection, intradermal injection, intrathecal injection, epiduralinjection, intraperitoneal injection, or intracerebral injection.Infusions are also contemplated by the methods of the invention (i.e.subdural, intrathecal or intracerebral infusion). If administration isintravenous, an injectable liquid suspension can be prepared andadministered by a continuous drip or as a bolus. In some instances, itmay be appropriate to administer the CFS compositions using an infusionpump.

The timing of administration of CFS compositions will depend upon thetype and severity of the disease, disorder or injury being treated. Inone embodiment, the CFS compositions are administered as soon aspossible after the diagnosis or injury. In another embodiment, CFScompositions are administered more than one time following diagnosis orinjury. In certain embodiments, where surgery is required, the CFScompositions are administered at surgery. In still other embodiments,the CFS compositions are administered at as well as after surgery. Suchpost-surgical administration may take the form of a singleadministration or multiple administrations.

CFS compositions may also be inserted into a delivery device, e.g., asyringe, in different forms. For example, the CFS compositions can bepart of a solution contained in such a delivery device. As used herein,the term “solution” includes a pharmaceutically acceptable carrier ordiluent. Pharmaceutically acceptable carriers and diluents includesaline, aqueous buffer solutions, solvents and/or dispersion media. Theuse of such carriers and diluents is well known in the art. The solutionis preferably sterile and fluid to the extent that easy syringabilityexists. Preferably, the solution is stable under the conditions ofmanufacture and storage and may optionally be preserved against thecontaminating action of microorganisms such as bacteria and fungithrough the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention canbe prepared by incorporating the CFS compositions in a pharmaceuticallyacceptable carrier or diluent and, as required, other ingredientsenumerated above.

CFS compositions may be administered systemically (for exampleintravenously), locally (for example by direct application undervisualization during surgery) or topically. For such administration, thecompositions may be in an injectable liquid suspension preparation or ina biocompatible medium which is injectable in liquid form and becomessemi-solid at the site of damaged tissue. A controllable endoscopicdelivery device can also be used.

Support matrices into which the CFS compositions can be incorporated orembedded include matrices which are recipient-compatible and whichdegrade into products which are not harmful to the recipient.

Natural and/or synthetic biodegradable matrices are examples of suchmatrices. Natural biodegradable matrices include plasma clots, e.g.,derived from a mammal, collagen, fibronectin, and laminin matrices.Suitable synthetic matrix material must be biocompatible to precludeimmunological complications. It must also be resorbable. The matrixshould be configurable into a variety of shapes and should havesufficient strength to prevent collapse upon implantation. Recentstudies indicate that the biodegradable polyester polymers made ofpolyglycolic acid fulfill all of these criteria (Vacanti, et al. J. Ped.Surg. 23:3-9 (1988); Cima, et al. Biotechnol. Bioeng. 38:145 (1991);Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 (1991)). Other syntheticbiodegradable support matrices include synthetic polymers such aspolyanhydrides, polyorthoesters, and polylactic acid. Further examplesof synthetic polymers and methods of incorporating or embeddingcompositions into these matrices are also known in the art. See e.g.,U.S. Pat. Nos. 4,298,002 and 5,308,701.

One of the advantages of a biodegradable polymeric matrix is that CFScompositions can be incorporated directly into the support matrix sothat it is slowly released as the support matrix degrades in vivo. Inaddition to the CFS compositions, other factors, including nutrients,growth factors, inducers of differentiation or de-differentiation (i.e.,causing differentiated cells to lose characteristics of differentiationand acquire characteristics such as proliferation and more generalfunction), products of secretion, immunomodulators, inhibitors ofinflammation, regression factors, biologically active compounds whichenhance or allow ingrowth of the lymphatic network or nerve fibers,hyaluronic acid, and drugs, which are known to those skilled in the artand commercially available with instructions as to what constitutes aneffective amount, from suppliers such as Collaborative Research, SigmaChemical Co., growth factors such as epidermal growth factor (EGF) andheparin binding epidermal growth factor like growth factor (HB-EGF),could be incorporated into the matrix or be provided in conjunction withthe matrix. Similarly, polymers containing peptides such as theattachment peptide RGD (Arg-Gly-Asp) can be synthesized for use informing matrices (see e.g. U.S. Pat. Nos. 4,988,621, 4,792,525,5,965,997, 4,879,237 and 4,789,734).

In another example, the CFS compositions may be incorporated in a gelmatrix (such as Gelfoam from Upjohn Company). A variety of encapsulationtechnologies have been developed (e.g. Lacy et al., Science 254:1782-84(1991); Sullivan et al., Science 252:718-712 (1991); WO 91/10470; WO91/10425; U.S. Pat. No. 5,837,234; U.S. Pat. No. 5,011,472; U.S. Pat.No. 4,892,538). During open surgical procedures involving directphysical access to diseased or damaged tissue, all of the describedforms of the CFS composition delivery preparations are availableoptions. These compositions can be repeatedly administered at intervalsuntil a desired therapeutic effect, i.e. accelerated wound healing, isachieved.

The three-dimensional matrices to be used are structural matrices thatprovide a scaffold to guide the process of tissue healing and formation.Scaffolds can take forms ranging from fibers, gels, fabrics, sponge-likesheets, and complex 3-D structures with pores and channels fabricatedusing complex Solid Free Form Fabrication (SFFF) approaches. As usedherein, the term “scaffold” means a three-dimensional (3D) structure(substrate and/or matrix). It may be composed of biological components,synthetic components or a combination of both. Further, it may benaturally constructed by cells or artificially constructed. In addition,the scaffold may contain components that have biological activity underappropriate conditions. The structure of the scaffold can include amesh, a sponge or can be formed from a hydrogel.

The design and construction of the scaffolding to form athree-dimensional matrix is of primary importance. The matrix should bea pliable, non-toxic, injectable porous template for vascular ingrowth.The pores should allow vascular ingrowth. These are generallyinterconnected pores in the range of between approximately 100 and 300microns, i.e., having an interstitial spacing between 100 and 300microns, although larger openings can be used. The matrix should beshaped to maximize surface area, to allow adequate diffusion ofnutrients, gases and growth factors. At the present time, a porousstructure that is relatively resistant to compression is preferred,although it has been demonstrated that even if one or two of thetypically six sides of the matrix are compressed, that the matrix isstill effective to yield tissue growth.

The polymeric matrix may be made flexible or rigid, depending on thedesired final form, structure and function. For repair of a defect, forexample, a flexible fibrous mat is cut to approximate the entire defectthen fitted to the surgically prepared defect as necessary duringimplantation. An advantage of using the fibrous matrices is the ease inreshaping and rearranging the structures at the time of implantation.

The invention also provides for the delivery of CFS compositions inconjunction with any of the above support matrices as well asamnion-derived membranes. Such membranes may be obtained as a by-productof the process described herein for the recovery of AMP cells, or byother methods, such as are described, for example, in U.S. Pat. No.6,326,019 which describes a method for making, storing and using asurgical graft from human amniotic membrane, US 2003/0235580 whichdescribes reconstituted and recombinant amniotic membranes for sustaineddelivery of therapeutic molecules, proteins or metabolites, to a site ina host, U.S. 2004/0181240, which describes an amniotic membrane coveringfor a tissue surface which may prevent adhesions, exclude bacteria orinhibit bacterial activity, or to promote healing or growth of tissue,and U.S. Pat. No. 4,361,552, which pertains to the preparation ofcross-linked amnion membranes and their use in methods for treatingburns and wounds. In accordance with the present invention, CFScompositions may be incorporated into such membranes.

Exemplary Therapeutic Uses of CFS Compositions Including, but notLimited to, ACCS, Pooled ACCS, PCS, SR-ACCS (including Pooled ACCS), andSR-PCS

Wound Healing

The CFS compositions of the present invention are effective inaccelerating wound healing of wounds caused by a number of sources,including but not limited to incisional, compression, thermal,radiation, penetrating, concussive, acute, chronic, infected, andsterile injuries. The instant invention is based upon the discovery thatCFS compositions can accelerate the wound healing process for all woundtypes, particularly when administered topically, i.e. to the surface ofthe wound site. Using CFS compositions all wound types, mechanical orthermal, acute or chronic, infected or sterile, undergo healing morerapidly than similar wounds left to heal naturally or which are treatedwith currently available methods. A “therapeutically effective amount”of a therapeutic agent within the meaning of the present invention willbe determined by a patient's attending physician or veterinarian. Suchamounts are readily ascertained by one of ordinary skill in the art andwill enable accelerated wound healing when administered in accordancewith the present invention. Factors which influence what atherapeutically effective amount will be include, the specific activityof the therapeutic agent being used, the wound type (mechanical orthermal, full or partial thickness, etc.), the size of the wound, thewound's depth (if full thickness), the absence or presence of infection,time elapsed since the injury's infliction, and the age, physicalcondition, existence of other disease states, and nutritional status ofthe patient. Additionally, other medication the patient may be receivingwill effect the determination of the therapeutically effective amount ofthe therapeutic agent to administer.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions and methods of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees centigrade, and pressure isat or near atmospheric.

Example 1 Preparation of AMP Cell Compositions

Recovery of AMP Cells

AMP cells were dissociated from starting amniotic membrane using thedissociation agents PXXIII, and trypsin. The average weight range of anamnion was 18-27 g. The number of cells recovered per g of amnion wasabout 10-15×10⁶ for dissociation with PXXIII and 5-8×10⁶ fordissociation with trypsin.

Method of obtaining selected AMP cells: Cells were plated immediatelyupon isolation from the amnion. After ˜2 days in culture non-adherentcells were removed and the adherent cells were kept. This attachment toa plastic tissue culture vessel is the selection method used to obtainthe desired population of AMP cells. Adherent and non-adherent AMP cellsappear to have a similar cell surface marker expression profile but theadherent cells have greater viability and are the desired population ofcells. Adherent AMP cells were cultured until they reached˜120,000-150,000 cells/cm². At this point, the cultures were confluent.Suitable cell cultures will reach this number of cells between ˜5-14days. Attaining this criterion is an indicator of the proliferativepotential of the AMP cells and cells that do not achieve this criterionare not selected for further analysis and use. Once the AMP cellsreached ˜120,000-150,000 cells/cm², they were collected andcryopreserved. This collection time point is called p0.

Example 2 Generation of ACCS

The AMP cells of the invention can be used to generate ACCS, includingpooled ACCS. The AMP cells were isolated as described above and ˜1×10⁶cells/mL were seeded into T75 flasks containing ˜10 mL culture medium.The cells were cultured until confluent, the medium was changed and ACCSwas collected 3 days post-confluence. Skilled artisans will recognizethat other embodiments for collecting ACCS from confluent cultures, suchas using other tissue culture vessels, including but not limited to cellfactories, flasks, hollow fibers, or suspension culture apparatus, etc.are also contemplated by the methods of the invention (see DetailedDescription above). It is also contemplated by the instant inventionthat the ACCS be cryopreserved, lyophilized or formulated forsustained-release following collection. It is also contemplated thatACCS be collected at different time point (see Detailed Description fordetails).

Example 3 Generation of Pooled ACCS

ACCS was obtained essentially as described above. In certainembodiments, ACCS was collected multiple times from an AMP cell culturederived from one placenta and these multiple ACCS collections werepooled together. Such pools are referred to as “SP pools” (more than oneACCS collection/one placenta). In another embodiment, AMP cell cultureswere derived from several placentas, i.e. from 5 or 10 placentas. TheAMP cells from each placenta were cultured and one ACCS collection fromeach culture was collected and then they were all pooled. These poolsare termed “MP1 pools” (one ACCS collection/placenta, multipleplacentas). In yet another embodiment, AMP cell cultures were derivedfrom several placentas, i.e. from 5 or 10 placentas. The AMP cells fromeach placenta were cultured and more than one ACCS collection wasperformed from each AMP cell culture and then pooled. These pools aretermed “MP2 pools” (more than one ACCS collection/placenta, multipleplacentas).

Example 4 Detection of Cytokines in Non-Pooled and Pooled ACCS UsingELISA

Standard ELISAs familiar to skilled artisan are performed on ACCS fromAMP cells obtained from 10 different placentas. In addition to assayingeach ACCS sample individually, pooled ACCS samples are also tested todetermine if variability of ELISA results between samples is reduced.ACCS is obtained as described above. Pools are made as follows: Pool 1is comprised of ACCS from placentas 1-5, Pool 2 is comprised of ACCSfrom placentas 6-10, and Pool 3 is comprised of ACCS from placentas1-10. In addition, ELISA of SP, MP1, MP2 pools is performed.

Example 5 Generation of PCS Compositions

The following PCS compositions are produced by combining the indicatedcytokine or factor at physiologic levels in a carrier:

Composition A: VEGF and TIMP-1

Composition B: VEGF, Angiogenin and TIMP-1

Composition C: VEGF, Angiogenin, PDGF-BB and TIMP-1

Composition D: VEGF, Angiogenin, PDGF-BB, TGFβ2 and TIMP-1

Composition E: VEGF and TIMP-2

Composition F: VEGF, Angiogenin and TIMP-2

Composition G: VEGF, Angiogenin, PDGF-BB and TIMP-2

Composition H: VEGF, Angiogenin, PDGF-BB, TGFβ2 and TIMP-2

Composition I: VEGF, TIMP-1 and TIMP-2

Composition J: VEGF, Angiogenin, TIMP-1 and TIMP-2

Composition K: VEGF, Angiogenin, PDGF-BB, TIMP-1 and TIMP-2

Composition L: VEGF, Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2

Composition M: Angiogenin and TIMP-1

Composition N: Angiogenin, PDGF-BB and TIMP-1

Composition O: Angiogenin, PDGF-BB, TGFβ2 and TIMP-1

Composition P: Angiogenin and TIMP-2

Composition Q: Angiogenin, PDGF-BB and TIMP-2

Composition R: Angiogenin, PDGF-BB, TGFβ2 and TIMP-2

Composition S: Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2

Composition T: PDGF-BB and TIMP-1

Composition U: PDGF-BB, TGFβ2 and TIMP-1

Composition V: PDGF-BB and TIMP-2

Composition W: PDGF-BB, TGFβ2 and TIMP-2

Composition X: PDGF-BB, TIMP-1 and TIMP-2

Composition Y: PDGF-BB, TGFβ2, TIMP-1 and TIMP-2

Compositions A-Y optionally contains Thymosin β4. Skilled artisans willrecognize that in certain embodiments other MMP inhibitors (i.e. TIMP-3,TIMP-4 or synthetic MMP inhibitors) may be suitable (J. FrederickWoessner, Jr., J. Clin. Invest. 108(6): 799-800 (2001); Brew, K., et al,Biochim Biophys Acta. 2000 Mar. 7; 1477(1-2):267-83).

VEGF, Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2 are added at thefollowing physiologic levels: ˜5-16 ng/mL for VEGF, ˜3.5-4.5 ng/mL forAngiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μgmL for TIMP-1 and ˜1.04 μg/mL for TIMP-2. VEGF may be obtained fromInvitrogen, catalog #PHG0144, PHG0145, PHG0146, PHG0141 or PHG0143;Angiogenin may be obtained from R&D Systems, catalog #265-AN-050 or265-AN-250; PDGF-BB may be obtained from Invitrogen, catalog #PHG0044,#PHG0045, #PHG0046, #PHG0041, #PHG0043; TGFβ2 may be obtained fromInvitrogen, catalog #PHG9114; TIMP-1 may be obtained from R&D Systems,catalog #970-TM-010; and TIMP-2 may be obtained from R&D Systems,catalog #971-TM-010. VEGF, Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2are added to a carrier such as normal saline, PBS, lactated Ringer'ssolution, cell culture media, water or other suitable aqueous solutionknown to skilled artisans.

The PCS compositions are tested in standard animal models for woundhealing to assess activity (see Definitions above for standard animalmodels for wound healing).

Example 6 Generation of Sustained-Release CFS Compositions

SR-CFS compositions, such as, for example, SR-ACCS (including pooledACCS) or SR-PCS, are produced by combining CFS compositions with any ofthe sustained-release formulation technologies described herein (seeDetailed Description) or otherwise familiar to skilled artisans.

Example 7 Effects of ACCS in an Animal Model of Chronic Wound Healing

An art-accepted animal model for chronic granulating wound was used tostudy the effects of ACCS on chronic wound healing (Hayward P G, RobsonM C: Animal models of wound contraction. In Barbul A, et al: Clinicaland Experimental Approaches to Dermal and Epidermal Repair: Normal andChronic Wounds. John Wiley & Sons, New York, 1990).

Results: ACCS was effective in not allowing proliferation of tissuebacterial bioburden. ACCS allowed accelerated healing of the granulatingwound significantly faster than the non-treated infected control groups.

Example 8 Use of CFS Compositions PCS, SR-ACCS and SR-PCS in an AnimalModel of Chronic Wound Healing

An art-accepted animal model for chronic granulating wound (Hayward P G,Robson M C: Animal models of wound contraction. In Barbul A, et al:Clinical and Experimental Approaches to Dermal and Epidermal Repair:Normal and Chronic Wounds. John Wiley & Sons, New York, 1990) is used tostudy the effects of the CFS compositions PCS, SR-ACCS or SR-PCS of theinvention on chronic wound healing.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Throughout the specification various publications have been referred to.It is intended that each publication be incorporated by reference in itsentirety into this specification.

1.-24. (canceled)
 25. A composition comprising a pooled amnion-derivedcellular cytokine solution (ACCS) comprising the proteins VEGF,Angiogenin, PDGF, TGFβ2, TIMP-1 and TIMP-2, wherein the proteins levelsare ˜5.0-16 ng/mL for VEGF, ˜3.5-4.5 ng/mL for Angiogenin, ˜100-165pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μg/mL for TIMP-1 and˜1.04 μg/mL for TIMP-2, the composition made by a method comprising thesteps of: a) obtaining a placenta and isolating the amnion from theplacenta, b) enzymatically releasing amnion-derived epithelial cellsfrom the amnion, c) collecting the released amnion-derived epithelialcells, and d) culturing the collected amnion-derived epithelial cells ofstep (c) in a tissue culture vessel for about 2 days in basal culturemedium that does not contain any non-human animal protein and is,optionally, further supplemented with recombinant human protein factorscapable of stimulating proliferation of the cultured amnion-derivedepithelial cells, e) selecting amnion-derived multipotent progenitor(AMP) cells from the amnion-derived epithelial cells of step (d) byremoving and discarding the culture medium which contains cells thathave not attached to the tissue culture vessel and keeping the cellsthat have attached to the tissue culture vessel, such attached cellsconsisting essentially of AMP cells, f) culturing the selected AMP cellsof step (e) in basal culture medium that does not contain any non-humananimal protein and is, optionally, further supplemented with recombinanthuman protein factors capable of stimulating proliferation of thecultured AMP cells until they reach confluence, g) removing the culturemedium from the confluent AMP cells in step (f) and culturing theconfluent AMP cells with fresh culture medium and culturing the AMPcells for 1, 2, 3, 4, 5, or 6 days, h) collecting the culture medium ofstep (g) to obtain ACCS and adding fresh medium to the confluent AMPcells, and i) repeating steps (g) and (h) a plurality of times andcombining the ACCS obtained in each step (h) to create the pooled ACCS.26. The composition of claim 25 wherein the AMP cells in step (g) arecultured in fresh culture medium for 3 days.
 27. The composition ofclaim 25 wherein the basal culture medium is supplemented with humanserum albumin.
 28. The composition of claim 27 wherein the human serumalbumin is up to concentrations of 10% human serum albumin.
 29. Thecomposition of claim 25 wherein the recombinant human protein factor isEGF.
 30. The composition of claim 29 wherein the EGF is at aconcentration of 10 ng/mL.
 31. The composition of claim 25 wherein thepooled ACCS is made using AMP cells derived from a single placenta. 32.The composition of claim 25 wherein the pooled ACCS is made using AMPcells derived from multiple placentas.
 33. The composition of claim 25which is further processed by the step of adding an agent capable ofeffecting sustained-release.
 34. The composition of claim 25 which isfurther processed by the step of lyophilization.
 35. The composition ofclaim 25 which is further processed by the step of formulating thecomposition for aerosolization.